Two-Stage Microparticle-Based Therapeutic Delivery System and Method

ABSTRACT

A system for delivery of a therapeutic agent to a site in mucosal tissue is provided. The system includes a porous, mucoadhesive polymeric matrix having a first and a second opposed surfaces. The matrix is formed by a composition including chitosan. The composition may also include any or all of a hydration promotor, a microparticle adhesion inhibitor, and a microparticle aggregation inhibitor. A plurality of microparticles having an average diameter between 500 nm and 2000 nm are embedded within the matrix. The microparticles contain a therapeutic agent and have a coating around the therapeutic agent. The first surface of the matrix is configured to be attached to the site in the mucosal tissue and the matrix is configured to provide controlled release of the microparticles through the first surface. The coating of the microparticles includes chitosan so as to provide controlled release of the agent from the microparticles.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.15/436,651, filed on Feb. 17, 2017, which claims the benefit of U.S.provisional applications: Ser. No. 62/336,405, filed on May 13, 2016;Ser. No. 62/296,599, filed on Feb. 18, 2016; and Ser. No. 62/336,209,filed on May 13, 2016. Each one of these applications is herebyincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants nos.R44CA192875-01 and number R44DE023725-03 awarded by The NationalInstitutes of Health. The government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates to systems for delivery of a therapeuticagent to a mucosal tissue, and in particular to systems with a porouspolymeric matrix including chitosan and a plurality of microparticlesembedded within the matrix.

BACKGROUND

Cancers affecting the mucosae of the body are a growing public healthconcern. Oral cancer alone affects over 640,000 people annuallyworldwide and over 40,000 in the US. The incidence is on the rise due toan increased affliction with oral HPV, which is cancer causing.Treatment methods include surgery and systemic chemotherapy administeredintravenously, often in combination. Surgery is often ineffective due tothe difficulty associated with identifying margins surrounding oraltumors. This inability to completely remove tumors during surgerycontributes to oral cancer's high rate of recurrence. Systemicchemotherapy is often used but lacks targeting and exposes the patient'sentire body to damaging chemotherapeutics. This method can be doselimiting due to exposure within the blood stream and other organs, asprecautions must be taken in consideration of the safety of thissystemic exposure. Systemic delivery often results in damaging sideeffects from toxic drugs reacting with the body. These includeneurotoxicity, nephrotoxicity, kidney failure, hair loss, nausea andmucositis.

In addition, oral cancer is among the most debilitating diseasesemotionally as well as physically. Permanent disfiguration can occurafter surgical resection of oral tumors. The patient's ability to eat,drink, or properly speak after surgery can also become impaired or notpossible. In part for these reasons, oral cancer is considered the mostexpensive cancer to treat. The costs associated with surgery andchemotherapy themselves are substantially high. However, in the case oforal cancer there also are significant costs required to reconstruct theface, neck or other regions affected by the large removal of tissue.These can include jawbone or oral tissue reconstruction. Further, theseprocedures can also leave the patient hospitalized in recovery for longperiods of time, which also contributes to substantial rehabilitativecosts post-operation. These costs can add up to a sum that has beenrecognized as the highest costs associated with cancer, and can exceedthe amount of US$150,000.

In addition to monetary cost and other physical side effects, emotionalside effects also speak to the especially tragic and debilitating effectof oral cancer compared to other cancers and diseases. The emotionaltoll on oral cancer patients can be far greater than that of otherdiseases, primarily due to the physical deformity (including physicalappearance and lack of clear speech) that results from treatment. Thepotential loss of significant portions of the tongue can be what leadsto permanently impaired speech and even taste. The severity of thisemotional effect can be fully understood when the suicide ratesassociated with other diseases are compared. The suicide rate ofpatients suffering from oral cancer is among the highest as compared toother cancers, and is about three times the rate of several other typesof cancer. These consequences of traditional treatment methods for oralcancer illustrate why an alternative treatment method is desperatelyneeded to address this unmet need and patient suffering.

Anal cancer accounts for 2.5 percentage of all digestive systemmalignances in the US, and approximately 8,000 new cases are diagnosedannually. The incidence of anal cancer in the general population hasincreased over the last 3 decades. Additionally, colorectal cancer (CRC)is a common and lethal disease. It is estimated that approximately134,490 new cases of large bowel cancer are diagnosed annually in theUS, including approximately 95,270 colon and 39,220 rectal cancers. Thiscancer remains the third most common cause of cancer death in the UnitedStates. Approximately 49,190 Americans are expected to die of largebowel cancer each year.

One of the differences between colorectal cancer and anal cancer are therisk factors that can cause each. Primary risk factors for colorectalcancer include age, genetics, race, diabetes, obesity, lack of exerciseand smoking. In contrast, the primary cause of anal cancer has been theincrease in prevalence of human papillomavirus (HPV).

As it pertains to anal cancer, almost all cases of anal cancer arecaused by HPV, which is cancer causing, the presence of the HPV genomehas been identified in 80%-85% of the cases of anal cancer. The HPV isable to live only in squamous epithelial cells that are found on thesurface of the skin and on moist surfaces—mucosal surfaces. The virus istransmitted through skin contact. Sexual activity and other skin contacthas been a primary driver of anal cancer. The primary driver of the risein incidence is due to the rise of HPV. Smoking is another risk factorfor anal cancer as it spreads carcinogens throughout the entire body andalso reduces the immune system's ability to fight the HPV virus. Over90% of anal cancer is squamous cell carcinoma, with the other 10%including more rare forms of cancer including basal cell carcinoma,adenocarcinoma, and malignant melanomas.

1. Anal Cancer Treatment:

Contrary to current perception, anal cancer is a very significant anddebilitating disease. Treatment can include surgery, radiation,chemotherapy, and combinations thereof, often resulting in significantside effects. Despite the various choices, these treatment optionsremain ineffective in many cases at eliminating tumors and preventingdeath. As stated in the background, the percentage of patients surviving5 years after treatment ranges between 45-85% according to the stage ofthe disease. The relatively low survival rate is unimpressiveconsidering that anal cancer is typically discovered at early stages.According to US government statistics, 49% of anal cancer is localizedat the time of diagnosis. An additional 31% exists regionally in thearea; fewer than 20% are node-positive, while only 13% is discoveredmetastasized. The low 5 year survival rate despite these localizedstatistics suggests that current treatment options are not optimal andmay be considered ineffective at treating anal cancer.

1.1 Surgery: While surgical treatment for anal cancer as the standard ofcare has since been replaced by chemotherapy, it is still used as ameans of tumor reduction when chemotherapy shows little effect. Thisradical procedure can require the removal of the anus, rectum, andsigmoid colon, with creation of a permanent colostomy. An ostomy as itpertains to anal and rectal cancer is a surgically created opening inthe abdominal wall that is used to divert bodily waste during and afteranal surgery. For anal cancer, the most common ostomy is the colostomy.Instead of expulsion via the anus, feces and other waste pass throughthe opening of this and into an external collection bag. A specializednurse is required to teach procedures regarding caring and washing ofthe ostomy and surrounding area. The process of surgically creating andpersonally maintaining the ostomy and collection bag can be verypainful, emotionally challenging, can result in infection, and can bevery expensive.

Developments of new strategies were directed at preservation of the analsphincter. Surgery has been associated with local failure in up to halfof cases, and five-year survival rates are approximately 50%-70%. In thepast, surgical treatment with abdomino-perineal resection (APR) (APR)was routinely performed for anal cancer. The radical procedure requiredremoval of the ano-rectum with creation of a permanent colostomy. Theoverall probability of five-year survival was 40-70%, with aperioperative mortality of 3%. When surgery is used, side effects areextensive and debilitating. The most common immediate complication (in32% of patients) is intra-abdominal or pelvic abscess, othercomplications include nerve injury (the autonomic nerves that affectboth sexual and urinary function may be injured), postoperative sexualor urinary dysfunction (10-60%), urologic injury, perineal wound, andcomplications related to the ostomy. Current treatment approachesreserve surgical therapy for patients with recurrent or persistentdisease after chemoradiotherapy. Although prognosis is poor overall, anAPR offers the potential for long-term survival. Local excision isperformed by several surgeons for small, local perianal cancer, wheresphincter function will not be compromised by adequate surgicalresection.

As mentioned before, in more serious (refractory or recurrent) cases ofanal cancer, APR is performed, in these cases, a permanent colostomy isneeded, and permanent damaging side effects are common. Failure tocontrol anal cancer and complications of treatment are alternativeindications for a colostomy, but in most cases, colostomy is requiredfor recurrent tumor.

1.2 Chemotherapy: Systemic chemotherapy is often used but lackstargeting and exposes the patient's entire body to damagingchemotherapeutics. This method can be dose limiting due to exposurewithin the blood stream and other organs, as precautions must be takenin consideration of the safety of this systemic exposure. Systemicdelivery often results in damaging side effects from toxic drugsreacting with the body. These include neurotoxicity, nephrotoxicity,kidney failure, hair loss, nausea and mucositis. As an alternative tosurgery, chemotherapy in addition to radiation are also used as methodsto treat anal tumors. The current standard of care uses initialconcurrent combination of chemotherapy and radiation for patients withanal canal squamous cell carcinoma, even with small, local tumors. Whenchemotherapy is used, temporary central venous catheters or peripherallyinserted central catheters may be used on an individual. Side effectsfrom treatment include those typical to systemic chemotherapy. Theseinclude nausea, hair loss, kidney damage, low blood cell count, mouthsores and a compromised immune system. Since chemotherapy is currentlydelivered systemically throughout the body, there are dose limitingfactors which can result in lower dosages being administered than whatis considered optimal.

1.3 Radiation: Forms of radiation administered include externalradiation or brachytherapy (internal radiotherapy, aiming to spare thesurrounding normal structures). These can be used as a treatment methodin combination with chemotherapy, and are very extensive. They arecommonly administered 5 days a week for 5 to 6 weeks. In addition to theside effects, this high frequency of administration contributes to thestrenuous nature of this treatment method. Side effects can persistpost-treatment and include irritation, pain during bowel movements andurination, vaginal pain or vaginal stenosis (for women) and erectiledysfunction or impotence (for men). Sexual and gastrointestinaldysfunction can occur and will often last throughout the remainder ofthe patients' life. The incidence of late toxicity from radiation suchas anal ulcers, stenosis, and necrosis, is also dose-dependent.

2. Rectal Cancer Treatment:

2.1. Surgery: Different surgical options are available, according to thestage, location, differentiation of the tumor. Superficially invasive,small rectal cancers managed with limited surgical procedure—trans-analexcision (TAE). Disadvantages of TAE alone are the high recurrence rate(up to 31%), potential compromise for cure, and the need for additionalwider resection when positive to cancer margins are found. Since themajority of patients have more deeply invasive tumors, more extensivesurgery is required, surgical options include total mesorectal excision(TME), low anterior resection (LAR) that includes rectum and sigmoidcolon removal.

These surgical options carry a variety of complications including riskof perioperative mortality. Wound infections, fecal frequency orurgency, the need for rectal reconstruction with its complications,anastomosis leak that leads to considerably high rates of morbidity andpossible mortality. Patients also risk functional derangements or, even,incontinence. Sexual and bladder function may also be adverselyaffected, probably because of injury to autonomic nerves.

The last surgical option is the abdominoperineal resection (APR) withits potential complications (mentioned above).

2.2 Radiotherapy: radiotherapy combined with chemotherapy can be used asa neoadjuvant (induction chemoradiotherapy) can be used for locallyadvanced or node-positive tumors, adjuvant treatment aims to improvelocal control and survival, and reduce recurrence.

2.3 Chemotherapy: As mentioned before, chemotherapy is used incombination with radiation as part of neoadjuvant or adjuvant treatment.The regimen of chemotherapy used is typically FOLFOX (5-FU, leucovorin,and oxaliplatin) or CapeOx (capecitabine and oxaliplatin). Thesechemotherapies are given systemically leading to systemic toxicities asmentioned prior.

Anal and rectal cancer together account for a very significant portionof cancer cases within the United States and worldwide, however due inpart to the side effects and limited efficacy described above, stilllack an ideal safe and effective treatment option. In order to addressthis severe unmet need, provided herein is an alternative, moreeffective treatment option for colorectal and anal diseases. Agentscommonly delivered in large doses systemically to treat these conditionshave been reformulated for localized delivery and retention, resultingin higher local concentration of agents while a lower overall dosage hasbeen administered. The present invention aims to overcome theshortcomings, side effects, and lack of efficacy of current treatmentoptions.

Difficulties in delivering therapeutic agents to mucosal tissues canalso be a hurdle to the treatment of diseases affecting thegastrointestinal (GI) tract. For example, over three million people arediagnosed with C. difficile colitis each year in the United Statesalone, and such disease can be caused by antibiotics which destroy thebody's natural bacteria. GI diseases, including inflammation, IBD andits variants can also stem from malnutrition, unsanitary livingconditions, exposure to bacteria, and other causes. These causes explaina significantly higher prevalence in developing nations, particularlythose in Africa and Southeast Asia. Children under five years of age areparticularly vulnerable to these conditions. There exists substantialrisk of life-threatening complications such as dehydration, sepsis,kidney failure, colon perforation, and death. These are diseases of avery debilitating, painful and widespread nature and represent achronically underserved market.

Despite the drawbacks of traditional drug administration routes, theyremain effective in certain cases at releasing therapeutic, diagnosticand/or prophylactic agents into the gastrointestinal tract. However,when employing these routes, local delivery of agents not only withinthe intestine but also through the intestinal mucosa remains verydifficult. The mucosa layer on the intestinal wall presents a formidablebarrier to adhesion and absorption of such agents, such as biologics,peptides, pharmaceuticals and nutraceuticals which are commonlydelivered in standalone forms. Few compositions can penetrate thisviscous, slippery material to reach the tissue and cells beneath.

Pertaining to targeted delivery, it is difficult for most substances tobecome attracted to these cells long enough to deliver an effectiveamount of the therapeutic, diagnostic and/or prophylactic agent. Thisremains one of the principal difficulties limiting the efficacy oftreatments delivered to the GI tract. Likewise, sufficient targeting toregions within the GI tract remains highly difficult due to the complexmakeup of the GI tract. Highly acidic levels within the stomach andvarying pH levels within different sectors of the intestine contributeto targeting difficulty, particularly to particle-based therapies. Forexample, the pH of the duodenum of a fasted patient can be approximately4.5 while the jejunum exhibits a pH closer to 6.5. A delivery systemwould in this example have to be able to withstand the stomach pH(1.5-2.5) in addition to the pH of the duodenum in order to be deliveredinto the jejunum. This scenario is present across the GI tract dependingon the region and fed vs. fasted condition of the patient.

SUMMARY OF THE EMBODIMENTS

In accordance with a first set of representative embodiments of theinvention, a system for delivery of a therapeutic agent to a site inmucosal tissue is provided. The system includes a porous, mucoadhesivepolymeric matrix having first and second opposed surfaces. The matrix isformed by a composition including chitosan, a hydration promoter, amicroparticle adhesion inhibitor, and a microparticle aggregationinhibitor. A plurality of microparticles are embedded within the matrix.The microparticles contain a therapeutic agent and have a coating aroundthe therapeutic agent. The first surface of the matrix is configured tobe attached to the site in the mucosal tissue and the matrix isconfigured to provide controlled release of the microparticles throughthe first surface. The coating of the microparticles includes chitosanso as to provide controlled release of the agent from themicroparticles. Optionally, the hydration promoter is selected from thegroup consisting of ethylene glycol, propylene glycol, beta-propyleneglycol, glycerol and combinations thereof. Also optionally, themicroparticle adhesion inhibitor is a non-ionic polymer, and, as afurther option, the non-ionic polymer is HPMC. Additionally, as anoption, the microparticle aggregation inhibitor is selected from thegroup consisting of monosaccharides, disaccharides, sugar alcohols,chlorinated monosaccharides, chlorinated disaccharides, and combinationsthereof. Also optionally, the microparticles further include sodiumtripolyphosphate. Optionally, in the system there is a free quantity ofthe therapeutic agent, embedded directly in the matrix, and nototherwise coated with chitosan, wherein the free quantity of thetherapeutic agent constitutes between 20-80% of a total quantity oftherapeutic agent in the system. Optionally, the second surface ispermeable to water. As a further option, the second surface includes amaterial selected from the group consisting of a polyacrylate adhesive,a non-woven polyester fabric, or combinations thereof. Optionally, thechitosan in the matrix and the chitosan in the microparticles is purechitosan. As a further option, the average diameter of themicroparticles is from about 500 nm to about 2000 nm.

In another embodiment, there is provided a porous, mucoadhesivepolymeric matrix formed by a composition including chitosan, a hydrationpromoter, a microparticle adhesion inhibitor, and a microparticleaggregation inhibitor. In accordance with yet another set ofrepresentative embodiments of the invention, there is provided a methodfor manufacturing a therapeutic agent delivery system. The methodincludes forming a first mixture with a plurality of microparticles. Themicroparticles contain a therapeutic agent and have a coating around thetherapeutic agent, the coating including chitosan. The method alsoincludes forming a second mixture from ingredients including the firstmixture, chitosan, a hydration promoter, a microparticle adhesioninhibitor, and a microparticle aggregation inhibitor. The method furtherincludes freezing the second mixture in a bath containing an aqueousalcoholic solution at a temperature above the freezing temperature ofthe aqueous alcoholic solution and at most −40° C., to form a frozenlayer precursor. Finally the method includes drying the frozen layerprecursor, to form a porous polymeric matrix with microparticlesembedded within the matrix. Optionally, the bath further contains dryice. Also optionally, the alcohol of the aqueous alcoholic solution isethanol. As a further option, the aqueous alcoholic solution is fromabout 90 wt % ethanol to about 99 wt % ethanol. Optionally, the methodfurther includes applying a second layer precursor to the frozen layerprecursor, to form a solid comprising a first layer and a second layer.Optionally, the second layer comprises a therapeutic agent. Alsooptionally, the wherein the drying is under vacuum.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of embodiments will be more readily understood byreference to the following detailed description, taken with reference tothe accompanying drawings, in which:

FIG. 1 is a graph showing the release profile of example particles whichmay be included within a multi-layered device according to embodimentsof the present invention.

FIG. 2 is an image of a multi-layered device according to embodiments ofthe present invention. The device features two layers: one top layerincluding FITC, a fluorescent dye which fluoresces green when examinedunder a fluorescent microscope, and one bottom layer including Cy5, afluorescent dye which fluoresces red when examined under a fluorescentmicroscope.

FIG. 3 represents another multi-layered delivery device according toembodiments of the present invention. The pictured also features twolayers: one “lighter” top layer containing 5-fluorouracil, achemotherapeutic, and one “darker” bottom layer containing cisplatin,another chemotherapeutic.

FIG. 4 is a microscope image taken from oral buccal tissue. The redlight is from the Cy5 fluorescent dye and the green light is from theFITC fluorescent dye.

FIGS. 5A-5C show three images taken from lamb buccal tissue at differenttime points. FIG. 5A shows fluorescent FITC and Cy5 above the tissueshortly after application of the device according to one of theembodiments of the present invention.

FIG. 5B shows FITC beginning to permeate the tissue after 30 minutes.FIG. 5C shows the green FITC permeating deeper into the tissue after 1hour.

FIG. 6 represents a multi-layer device according to embodiments of thepresent invention included as part of a treatment kit for use inmucosa-based indications according to embodiments of the presentinvention.

FIG. 7 is a schematic depiction of a kit containing 6 treatment devicesaccording to embodiments of the present invention.

FIG. 8 is an image taken of one of the treatment kits designed inaccordance with the depiction of FIG. 7.

FIG. 9 is another schematic representation of a kit according toembodiments of the present invention.

FIG. 10: Illustration of the human GI tract from the stomach onwards andassociated pH levels of each region.

FIG. 11: Depiction of how solutions containing particles (“Ps”) withdifferent properties can be combined to offer targeting to multipleexclusive regions within the GI tract according to embodiments of thepresent invention.

FIG. 12: Graphical depiction of epithelial cells and the location ofcrypts, which are one of the targeted areas the disclosed concept.

FIG. 13: A depiction of a particle containing an active agent accordingto embodiments of the present invention. As the image illustrates,targeting ligands, crosslinkers, agents, additional polymers andcombinations may be included within the particles.

FIG. 14: ATTO (Red fluorescent dye) labelled particles according toembodiments of the present invention permeating into and beyond thebasement membrane of intestinal mucosa.

FIG. 15: Permeation of ATTO (fluorescent dye) labelled particles (red)permeating into intestinal tissue beyond the basement membrane accordingto embodiments of the present invention. Depth of approximately 500 μmis evident. 100× magnification.

FIG. 16: A graphic chart illustrating the delivery and absorptionprocess according to embodiments of the present invention.

FIG. 17: Illustration depicting how, according to embodiments of thepresent invention, enteric capsules may be used to target intestinalstem cells.

FIGS. 18A-18C: Data from a cell uptake study, including a microscopyimage 1 hour after incubation with Alexa 647 particles (FIG. 18A).Evaluation of cell uptake by flow cytometry, percentage of positivecells after 30 and 60 min incubation with particles at a concentrationof 0.03 g/L (FIG. 18B). The results showed that particles were taken upby more than 95% of the cells in both cases FIG. 18C: Fluorescenceintensity was 23% higher after 60 min incubation vs. after 30 min due tohigher NP uptake.

FIG. 19: Image showing how the design of enteric capsules would includeand protect particles.

FIG. 20: Image showing an example of the relationship of pH to size ofparticles in some embodiments of the present invention.

FIG. 21: Image showing an example of the relationship of pH to PDI(defined below) of particles in some embodiments of the presentinvention.

FIG. 22A is a schematic drawing of a particle according to an embodimentof the present invention. FIG. 22B includes a list of parameters whichmay be modified to control the degradation of particles according toembodiments of the present application.

FIG. 22C illustrates release of encapsulated agents from particles asthe pH increases.

FIG. 23A: synthesis pH size vs. pH adjustment; the particles (“NPs”)synthesized at pHs ranging from 1.0-6.0 showed a consistent trend in thepH of their disintegration. Nearly all of the NPs began to swell anddegrade once the increased pH was a factor of 2 or more than theirsynthesis pH. FIG. 23B: pH change to size; results show that even if NPsare synthesized at a pH of 5, for example, they can remain stable in avery acidic environment where they will not release their encapsulateddrug. NPs are considered to begin releasing their payload once theirsize increases 400% from that of initial synthesis.

FIG. 23C: pH change to polydispersity index (PDI). The PDI is a keyfactor of NPs stability. A PDI of 1 signifies that the solution isincredibly polydisperse, indicating that particles have fallen apart.

FIGS. 24A-24D: Graphs illustrating the sensitivity of one selectedloaded particle (“LP”) variant (FIGS. 24A and 24B) as well as graphsillustrating the level of modulation and control that can be held overparticle properties (FIGS. 24C and 24D) according to embodiments of thepresent invention. FIGS. 24A and 24B illustrate the sensitivity of LPssynthesized from chitosan chloride rather than the raw chitosan polymer.LPs made from chitosan chloride proved to be more fragile and sensitiveto pH changes than the raw counterpart. FIGS. 24C and 24D illustrate thelevel of modulation and control that can be held over LP properties fromchitosans of different compositions and molecular weights using sodiumnitrite.

FIGS. 25A-25D: Charts demonstrating an example of how the charge (FIG.25A) and size (FIGS. 25B, 25C, and 25D) of chitosan loaded particles canbe precisely modulated. In FIGS. 25A and 25B is plotted the effect ofdifferent pre-processing time in relation to NP charge and sizerespectively. In FIGS. 25C and 25D are graphed the effects of usingdifferent concentrations of a preprocessing agent such as sodiumnitrite.

FIGS. 26A-26E: Tables showing the polydispersity (PDI) and average sizein nanometers of particles synthesized at various pH levels which havebeen designed to remain stable at desired secondary pH levels.

FIG. 27: Illustration demonstrating how particles can be created torelease at specific desired regions within the GI tract according toembodiments of the present invention.

FIG. 28 illustrates a mesh containing microparticles for unidirectionaldelivery through either the mucosa or epithelium lining the colon,rectum or anal tissue as outlined in the description deliveringmicroparticles into the tissue, according to embodiments of the presentinvention. The mesh depicted contains an optionally impermeable or waterpermeable backing layer to prevent passage of materials through thereverse side of the mesh, and illustrates how loaded particles releasedpermeate the tissue locally.

FIG. 29 is an image of one style of the mesh according to embodiments ofthe present invention within a protective mold.

FIG. 30 is an image showing the fluorescent permeation of one style ofthe mesh within tissue. The mesh was administered and left in contactwith the tissue for one hour. particles have been conjugated to ATTO(fluorescent dye) which fluoresces red as shown in the image. The bluecolor is DAPI staining which stains the nuclei of cells blue. The scalebar shown is 400 μm, showing that the red particles have permeatedsignificantly into the blue tissue.

FIG. 31 shows a graph detailing the stability of particles in varioussolutions.

FIG. 32 shows the mesh containing particles inside of it according toembodiments of the present invention.

FIG. 33 shows the level of permeation by one embodiment of the presentinvention. The green fluorescence represents particles which havepermeated the tissue.

FIG. 34 is a chart comparing permeation and release of chitosanparticles from a patch having a chitosan matrix in the presence (+PG) orabsence (−PG) of propylene glycol according to embodiments of thepresent invention.

FIGS. 35A-35B show chromatograms monitoring the release of5-Fluorouracil (“5-FU”) from a device including particles loaded with5-FU in a chitosan matrix according to embodiments of the presentinvention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Definitions. As used in this description and the accompanying claims,the following terms shall have the meanings indicated, unless thecontext otherwise requires:

A “polymer” is a molecule having at least 100 units of a monomer.

“Microparticles” are sets of particles having an average diameter of atleast 200 nm to at most 2000 nm.

“Nanoparticles” are sets of particles having an average diameter of atleast 1 nm to below 200 nm.

A “particle diameter” or “particle size” is the length of the longeststraight axis between two points on the surface of the particle.

A “pure chitosan” is a chitosan that is not a salt of chitosan.

A “microparticle adhesion inhibitor” is an additive that lowers theattractive forces between a polymeric matrix and particles embeddedtherein. As a result, the particles can move through the matrix at afaster rate than in the absence of the adhesion inhibitor.

A “microparticle aggregation inhibitor” is an additive that lowers thetendency of particles embedded in a matrix to aggregate when the matrixis subjected to freezing. As a result, the particles are less likely tosuffer from damage or destruction when the freezing takes place.

A “mucoadhesive” material is characterized as having the ability toadhere to mucosal membranes in the human body.

A matrix is “porous” when a fraction of its volume is void space. Insome instances, the void space is accessible from the outer surface ofthe matrix, so that items present in the void space, such asmicroparticles, may migrate to and from the outer surface.

“Mucosal tissue” is tissue having an associated mucosa. In particular,mucosal tissue includes the mucosa and also tissue underlying themucosa. A “site in mucosal tissue”, where, for example, a canceroustumor is present may involve not only the mucosa but also tissueunderlying the mucosa.

“Polydispersity index” (PDI) or simply, “dispersity” is a measure of theheterogeneity of sizes of a set of particles, for example microparticlesin a mixture.

“Zeta potential” (ZP) is a measure of the overall charge that a particleacquires in a particular medium. The ZP may be measured on a ZetasizerNano instrument.

“Permeation” is the ability to pass through or penetrate, a mucosa, itsunderlying tissue, or both.

“Biocompatible” refers to the ability of a biomaterial to perform itsdesired function with respect to a medical therapy, without elicitingany significant undesirable local or systemic effects in the recipientor beneficiary of that therapy, but generating the most appropriatebeneficial cellular or tissue response in that specific situation, andoptimizing the clinically relevant performance of that therapy.

“HPMC” refers to hydroxypropyl methylcellulose, also known ashypromellose. “Biodegradable” refers to a property of the materials thatis capable of being broken down especially into innocuous products bythe action of living things.

“Kilo count per second” (Kcps)”, mean count rate (in kilo counts persecond (kcps)). For example, the threshold may be set such that when thecount rate of the sample is lower than 100, the measurement should beaborted, meaning the concentration of the sample is too low formeasurements. A sample with suitable Kcps can be considered a stablesample with idea concentration for measurement.

“Mesh” refers to a device, sponge, wafer, or like product which containselements incorporated within it to be released from the mesh when it isapplied to a mucosa.

A “system for delivery of a therapeutic agent based on a polymericmatrix and microparticles” may also be referred to as an “agent deliverydevice” or as a “delivery patch”.

Unless otherwise specified, the term “wt %” refers to the amount of acomponent of a system for delivery of a therapeutic agent, as expressedin percentage by weight.

Unless otherwise specified, the “molar mass” of a polymer is intended tomean the number average molar mass of the polymer molecules.

Improved Matrix and Particle Device

In a first aspect, the present application provides improvements to thetechnology described in published U.S. application number US2014/0234212, entitled “Targeted Buccal Delivery of Agents”, which ishereby incorporated herein by reference in its entirety.

In a first set of representative embodiments, there are provided systemsfor delivery of a therapeutic agent based on a polymeric matrix andmicroparticles which are improved by the addition of a hydrationpromoter to the matrix. Example hydration promoters include hygroscopiccompounds such as glycols, for instance ethylene glycol, propyleneglycol, beta-propylene glycol, and glycerol. Exemplary concentrationranges for the amount of hydration promoter include from about 0.001 toabout 10 wt %, from about 0.01 to about 5 wt %, and from about 0.1 toabout 1 wt %.

Without wishing to be bound to any particular theory, it is believedthat the hydration promoter increases moisture absorption by thedelivery system. This increase in hydration enables the rapid releaseand permeation of the microparticles from the matrix. It is alsobelieved that the hydration promoter improves uniformity and durabilityby acting as a cryoprotectant during the manufacturing process of thedelivery system. Again without being bound to any particular theory, itis believed that the hydration promoter acts as a “spacer” between icecrystals and matrix polymer molecules, to ensure a uniform freezingpattern. The resulting structure is more flexible, uniform, and durablethan in the absence of the hydration promoter.

To illustrate the improvement in performance imparted by hydrationpromoters, patches including a chitosan polymer matrix and chitosanmicroparticles were manufactured with and without propylene glycol (PG)in the matrix. The particle release and permeation of the patches wasmeasured for both types of patch, and the experiment was run intriplicate. As reported in the chart of FIG. 34, the average percentageof permeation in the presence of propylene glycol ((+)PG) was 94% with astandard deviation of about 3%, which dropped to 43% with a standarddeviation of 27% in the absence of propylene glycol ((−)PG). Thepercentage of release in the presence of propylene glycol was 95% with astandard deviation of about 3%, as against 21% with a standard deviationof 41% without PG. Clearly, patches with PG performed markedly betterthan those without, and release and permeation numbers were morereproducible as shown by the smaller standard deviations.

In another set of representative embodiments, there are provideddelivery devices improved by the addition of an adhesion inhibitor.Without wishing to be bound to any particular theory, it is believedthat when the matrix and particles are made of materials bearing polaror ionically charged moieties, such as chitosan, the mobility of theparticles suffers. In the instance of chitosan, it is believed that theinteractions between acetyl and amine moieties of the polymer cause theparticles to adhere to the matrix and inhibit their release.

It has been found that the inclusion of an adhesion inhibitor canmitigate adhesion of the matrix with the particles. Without being boundto any particular theory, it is believed that the adhesion inhibitoracts as a “spacer” between the chitosan of the particles and thechitosan in the body of the matrix, releasing the particles and allowingfor improved drug release profiles. Representative example adhesioninhibitors include non-ionic polymers such as hydroxypropylmethylcellulose (HPMC). Depending on the application, the molar mass ofthe non-ionic polymer may be from about 1 kDa to about 200,000 kDa,while its viscosity may vary from about 10 cps to 100,000 cps. Inrepresentative embodiments, the molar mass of the non-ionic polymer isfrom about 10 kDa to 30 kDa, and its viscosity from about 10 cps toabout 100 cps. Depending on the application, the amount of adhesioninhibitor may be from about 0.1 wt % to about 99 wt %. In someembodiments, the amount of adhesion inhibitor is from about 0.1 wt % toabout 25 wt %.

In a further set of representative embodiments, delivery devicesimproved by the addition of an aggregation inhibitor are disclosed.Processes for manufacturing the delivery devices include freezing stepsduring which ice crystals may form within the matrix. Such crystals canforce the microparticles into each other, creating particle aggregateswhere the particles are damaged or destroyed. Again without wishing tobe bound to any particular theory, it is believed that aggregationinhibitors exert a cryoprotectant action by forming crystalmicrostructures which prevent aggregation of the particles.Carbohydrates and carbohydrate derivatives provide exemplary types ofaggregation inhibitors, including monosaccharides, disaccharides, sugaralcohols, chlorinated monosaccharides, and chlorinated disaccharidessuch as sucralose. Depending on the application, the amount ofaggregation inhibitor in the patch may be in the range from about 0.1 toabout 50 wt %. In some embodiments, the amount of aggregation inhibitoris from about 1 to about 10 wt %.

In another set of representative embodiments, improved pure chitosanmicroparticles are provided. Traditional chitosan particles aremanufactured with salts of chitosan characterized by a high degree ofdeacetylation and bearing electrically charged moieties, for examplechitosan chloride and chitosan glutamate. It has been found that betterresults are provided if the particles are made from pure chitosan, amaterial characterized by not being a salt, that is, with its aminegroups unprotonated, and having a degree of deacetylation of at least70%. In particular, the particles are characterized by larger diametersthan traditional particles. In some embodiments, the average diameter ofthe pure chitosan particles may range from about 200 to about 2000nanometers. In other embodiments, the average diameter ranges from about500 to about 2000 nanometers, and in additional embodiments from 500 to1000 nm.

In a further improvement, chitosan microparticles improved by theaddition of sodium tripolyphosphate (STPP) are provided. Without wishingto be bound to any particular theory, it is believed that the STPPfunctions as a cross-linker to form the particles by acting as anegative counter-ion to the positively charged amine groups on chitosan.This electrostatic interaction forms ionic bonds that support thestructure of the particles. Also without wishing to be bound to anyparticular theory, it is believed that the presence of sodium aspositive counterion renders STPP a more effective crosslinker than otherTPP salts.

It has also been found that when the matrix includes a free quantity ofthe therapeutic agent, embedded directly in the matrix and not otherwisecoated with chitosan in the particles, the device is therapeuticallymore effective than comparable matrices which include either only a freequantity of the therapeutic agent or only therapeutic agent coated withchitosan. In representative embodiments, the free quantity of thetherapeutic agent constitutes between 20-80% of the total quantity oftherapeutic agent in the delivery system.

FIG. 1 is a graph showing the release profile of example particleshaving an average diameter in the range of 500 to 2000 nm. The graphshows the rate of release of the encapsulated agent from thenanoparticles over 60 hours. Cisplatin was used for this experiment dueto its use in the treatment of oral cancer and its ease of detection viaatomic absorption spectrometry (AAS). Cisplatin is platinum-based, andAAS can detect amounts of platinum as small as 5 μg/L. The graph shows45% of cisplatin released from NPs over 60 hours.

FIGS. 35A-35B show chromatograms monitoring the release of5-Fluorouracil (“5-FU”) from a device including particles loaded with5-FU and embedded in a chitosan matrix. The particles and matrix, theparticles having an average diameter in the range of 500 to 2000 nm,were made with the same procedure and ingredients as the device of FIG.1, except that this time the particles contained 5-FU instead ofcisplatin. The particles where embedded within the matrix to form aparticle/matrix device, and the release of 5-FU was monitored via HPLC.The peak at 1.968 is acetic acid contained within the device. Bothacetic acid and 5-FU were detectable with ultraviolet light at the 265nm wavelength emitted by the detector.

In example systems for delivering a therapeutic agent to a site in amucosal tissue, the matrix has a first and second opposing surfaces. Thefirst surface is configured to be attached to the site in the mucosaltissue, and the matrix is configured to provide controlled release ofthe microparticles through the first surface. It has been found thatrelease of the microparticles is improved if the second surface ispermeable to water. Example water-permeable coatings which may beapplied to the second surface include polyacrylate adhesives andnon-woven polyester fabrics.

Localized Delivery of Agents Via a Multi-Layered Delivery Device

In one aspect, one or more of the above improvements may be applied to amulti-layered agent delivery device. The multi-layered device is capableof delivering the same or multiple agents in phases over a period oftime or delivering multiple agents concurrently via modulation of themakeup of each layer. The device and a method for manufacturing suchdevice have been developed to address the unmet need of deliveringagents in a multitude of forms locally to mucosal tissue. The multiplelayers within this platform may be used for varying purposes.

Traditional drug delivery to a mucosa consists of an initial bolus doseof agent followed by a steady reduction in exposure over time. Thisplatform is able to mitigate this tendency via its multiple layers andthe inclusion of microparticle within at least one layer. The materialforming the structure of each layer can be optionally chosen to degradeslowly, and the same agent (such as cisplatin for the local treatment ofa cancerous tumor) may be chosen for inclusion within each of themultiple layers.

In one example embodiment, the device therefore can be designed torelease cisplatin locally in multiple phases, providing significantlylonger treatment without the side effects, multiple doses required ordose limiting hindrances associated with cisplatin that is parenterallyadministered. The inclusion of microparticles within this device furtherassists in the device's ability to provide a sustained local dosage. Themicroparticles included within this device are released once it isapplied, permeate the mucosal tissue, and remain local within the tissuebeneath which the device was applied. These microparticles then degradeover a period of time, further providing a sustained, longer dosage ofagents. When different agents are included within each layer, additionalobjectives are able to be achieved. For example, if the device isapplied for the treatment of a recently acquired open wound, a painmitigator and anti-infective agent can each be included within a layer.

When applied within the oral cavity, the device is placed directly ontoaffected oral tissue within the mouth and releases agents for controlledand targeted treatment of oral diseases. Agents which may be included infree form (such as a pain mitigator in the first layer) may be designedto have an immediate effect to the underlying tissue, whereas agentsencapsulated within microparticles (such as a chemotherapeuticpharmaceutical) may be included within a second or subsequent layer. Themicroparticles are then able to act independent of the first agent,permeate the underlying tissue, and provide a sustained, longer termdelivery of agent to the tissue. This device overcomes deficiencies ofother prior art by offering the ability to modulate the duration andtreatment order parameters to provide multiple stages and durations oftreatment.

In addition, the device may be further included within a treatment kitto optimize its safety and efficacy. The kit can be optimized for amucosa. FIG. 6 represents an example multi-layer device included as partof a treatment kit for use in mucosa-based indications. The figureillustrates a kit containing six treatment devices, where: 61 refers toa wetting agent or permeation enhancer which may be externally appliedto the mucosa prior to treatment (in either solution or powder form), 62refers to gauze or other similar absorbent pad which may be used tomoisten and keep the device in place within the oral cavity, 63 a bagfor disposal of hazardous waste (for use when hazardous agents areincluded or otherwise), 64 identifies forceps or forceps-like devicesused to keep the device in place and to prevent swallowing, and 65refers to six individually packaged and stored treatment devices.

The release of agent(s) from the device is activated in part by exposureto moisture. Therefore, a moisturizing solution such as saline may beprovided with the device to be used during the application process.Further, permeation enhancers in powder or solution form may be includedto be externally applied to the mucosa prior to application of thedevice. The permeation enhancer may optionally be included in the formof a powder which requires reconstitution. The powdered form may beincluded to maintain stability of the permeation enhancer. When includedin this form, the kit may optionally include additional materials amongwhich at least one glass vial (5 mL to 20 mL in size) containing sterilewater to be used for reconstitution. The kit may additionally includesyringes (such as 3 mL Luer-lock syringes) and aspirating needles (suchas 18 G needles) to be used for reconstitution of the permeationenhancer.

In addition, when the device is used for certain indications (such asoral indications), care must be taken to ensure that the product issafely applied and removed to prevent choking or swallowing. The kitdisclosed herein addresses these concerns by including all materialsnecessary to ensure the safe application device. Example kits include atleast one pair of forceps (either multi-use metal forceps or single usedisposable plastic forceps) or other similar instrument used to positionand place the device to prevent exposure of agents to people or exposureof the device to the throat.

Disposable packaging can also be included within the kit to ensure thesafe disposal and non-contamination of the treatment process byisolating the materials used during treatment. This packaging mayinclude a hazardous waste package used when toxic drugs such as thoseused to treat oral cancer or melanoma are administered, or biohazardpackaging. Additionally, empty scintillation vials may be included tocollect the used device post-treatment for purposes such asresidual-agent analysis.

In representative embodiments, a method of manufacturing of amulti-layered device and a formulation created according to such methodare provided, as shown in the non-limiting examples of FIG. 2 and FIG.3). The method includes the freezing and freeze drying of polymericsolutions containing a therapeutic agent.

Precursor mixtures are first created, then subjected to freezing orfreeze drying. The device may feature multiple layers, as illustratedfor instance in FIG. 2 and FIG. 3, and the precursor mixture to eachlayer may be separately made. All layers may each contain anindependently chosen agent to be delivered, and at least one layercontains microparticles which further encapsulate at least one of theagents. The microparticles may be synthesized, for instance, accordingto the ionotropic gelation method, where no modification of the agenttakes place. Microparticles are designed to range from 200 to 2000nanometers, more preferably 500 to 2000 nanometers, and yet morepreferably from 500 to 1000 nm in average diameter. Agents such as apermeation enhancer, taste masking elements and agents for the formationof body structure may be added. These agents may include propyleneglycol, hydroxypropylmethylcellulose, chitosan, sweeteners, peppermintor other flavorings, among many others. Solutions containing agents butno microparticles may also contain these and other agents.

Once ready, the precursor mixtures are subjected to freezing. It ispreferable that the layers of the device be first frozen in a freezingbath of an aqueous alcohol at a temperature of at most −40° C., forexample in a bath of aqueous ethanol and dry ice. This method has beenfound to result in a device which is able to release nearly all of itsagent content and permeate deeply into the desired mucosal depths.Without wishing to be bound to any particular theory, the product deviceis more effective as compared to other methods of freezing. When theprecursor mixture was frozen via liquid nitrogen, placement in a freezerat −80° C., or standalone dry ice, some of the microparticles burst andthe polymer in the matrix of the device became more rigid, resulting ina low percentage of agent release and a compromised therapeuticefficacy. For best results, the bath should include dry ice completelycovered by a solution of at least 90 wt % ethanol in water. Theprecursor mixture of the first layer of the device (in liquid form) ispoured into a mold, for example a silicone molding and is submergedapproximately ⅔ to ¾ in the bath of ethanol and dry ice, to form afrozen layer. Preferably, thirty minutes should be allowed to achievecomplete freezing.

After freezing the initial layer, a second layer may be added by one oftwo methods. In the first method, the precursor mixture of the secondlayer is poured in liquid form on top of the frozen first layer whilethe first layer remains in the ethanol/dry ice bath. The resultingfrozen bottom layer and liquid top layer are then submerged more deeplyuntil a ⅔ to ¾ overall submersion ratio is met. Another 30 minutes areallowed for complete freezing of the second layer. Subsequent layers inexcess of two may be added by the same process.

In the second method, each layer is separately and concurrently frozenin its individual mold within the freezing bath. After thirty minutesare allowed to ensure complete freezing of each layer, a coating of ansolution of one or more salts, for example 0.12% saline, is brushed ontothe first, initial layer. Within about a minute of the application ofthe coating, the second layer is applied onto the first and a pressureof about 0.25 kg is applied.

This results in a combined solid. Subsequent layers in excess of thesecond layer can be applied by the same method.

After all the desired layers have been added, the device is moved into alyophilization chamber for about one to three days, depending on thenumber of devices loaded into the chamber. After the lyophilizationremoves all liquids, the multi-layer device is ready for use, asillustrated in the examples of FIG. 2 and FIG. 3.

In one representative embodiment, a multi-layered device may be used forthe delivery of multiple agents over a concurrent period of time. Forexample, if use for the treatment and pain mitigation of mucositis isdesired, one layer may include a pain mitigator, and one layer mayinclude an agent for the treatment of mucositis.

In another representative embodiment, a multi-layered device may be usedfor the delivery of multiple agents over a prolonged period of time(FIG. 4, FIG. 5). If the example of mucositis is again used, multiplelayers may include a pain mitigator in free form, a pain mitigatorencapsulated within microparticles and an agent for the treatment ofmucositis encapsulated within microparticles. The initial freeform layeris able to provide immediate pain relief, and the subsequentparticle-encapsulated layers are capable of delivering microparticlesbeneath the tissue, where they further release their agents over aperiod of days, providing longer-term pain relief and treatment. FIG. 4and FIG. 5 illustrate this effect. Fluorescence was used in order toprovide detection. As shown, the multiple fluorescent layers releasedover time and permeated the tissue at different rates, providing acustomized treatment.

Kit for the Treatment of Oral Cancer and Other Oral Diseases

In another aspect, a kit, including a local drug delivery device withinthe mouth and materials for its successful administration and subsequentdisposal is provided. This kit is specifically designed to provide theproper tools and treatment device required to deliver agents into oraltissue to treat oral diseases. Targeted delivery within the mouth intothe oral tissue is desired. The motivation behind the development ofthis kit has been to treat the severely underserved market pertaining tountreated or poorly treated oral diseases.

One of the preferred embodiments of the kit delivers a safer and moreeffective oral cancer treatment locally into the oral tissue. In thisembodiment, the active drug is included within a treatment device, whichis in turn included in the present kit. The kit also includes a numberof elements to ensure a safe treatment process for both clinicians andpatients, such as thorough proper handling, disposal, and storage of thetreatment device, as well as at least one permeation enhancer. At leasta fraction of the active drug within the treatment device isencapsulated within mucoadhesive microparticles, which leads to localretention of the drug within the oral tissue. As a result, only afraction of the chemotherapeutic is required, but the local targetingenables a more concentrated dosage at the tumor location compared to alltraditional treatment options. An application via this kit can result inthe significant reduction of side effects, more effective treatment,elimination of debilitating surgery and recovery, and a safer overalltreatment.

The improvements of this kit over traditional treatment devices are inpart based upon the administration, disposal, storage, and/or packagingrequirements of the treatment device. In certain embodiments, highlysensitive and potent chemotherapeutics are contained within thetreatment device. Proper handling procedures must be followed to preventexposure of the chemotherapeutic to clinicians, or improper applicationto patients. Materials which set out these proper handling proceduresmay be included with the kit. Proper disposal of the treatment devicefollowing administration may be essential to prevent potentialcontamination or exposure. Procedures to wash the treatment locationwithin the mouth both before and following administration may benecessary and therefore proper materials for that purpose must also besupplied. The treatment device must also be packaged appropriately asmany chemotherapeutics and the treatment device are sensitive to lightand humidity. Packaging must be included for the device and willpreferably be made of pharmaceutical grade materials. There may also bea small protective insert of plastic where the wafer is held within asmall plastic cup sealed in a pouch. There may also be materials such asLuer-lock syringes, aspirating needles and sterile water to be used forreconstitution of the permeation enhancer (if provided in powder form).

These improvements are conducive to forward the commercialization of thedevice and kit disclosed herein. The improvements also protect thesafety of operators from the agents included within the treatment deviceand the patient from improper administration, which could result in theaccidental swallowing of the device. The use of additional componentswithin the kit also facilitates higher compliance rates among patients,and consequently a greater number of successful treatments. Properpreparation and cleaning of the application area will also preventirritation to the oral tissue.

Oral mucositis is also a significant disease due in part to the factthat it can occur in the mouth when systemic chemotherapy is given forany reason, not only oral cancer. In a second embodiment, the treatmentdevice within the disclosed kit may include agents which treat orrelieve pain, or otherwise address oral mucositis. Unlike existingtreatments for mucositis, the current kit includes a device whichcontains agent-encapsulated microparticles within it. In a mannersimilar to its effect treating oral cancer, the microparticles releasedfrom the device are mucoadhesive so that they remain local to the siteof the mucositis. Since the particles are nanoscale, they are able topermeate the tissue and release the encapsulated agent deeper within theaffected area than other current treatments, and without broaderexposure of the agent. Administration via this kit is able to offer amuch more effective delivery of agents to areas affected by mucositis.

In another embodiment, the described kit is also used to deliver agentsfor the treatment of precancerous/premalignant oral lesions.Precancerous/Premalignant lesions are often left untreated when detecteddue to a lack of ideal treatment options. A diagnosis is often coupledwith monitoring for malignancy rather than early treatment becausechemotherapy or surgery can be considered too extreme for early lesions.It is also difficult to differentiate between precancerous/premalignantlesions and other non-malignant lesions. For this reason, there oftenexists an unwillingness to administer damaging systemic chemotherapy fora potentially non-life threatening issue. To address these conditions,the present kit can be used to administer lower dosages ofchemotherapeutics or other agents to these lesions. Treatment would beable to be administered on a much larger scale due to the higherefficacy achieved with such a small dosage and the significantly highersafety. For these reasons, the present kit is viewed as a significantimprovement and viable alternative to current treatment methods.Furthermore, the inclusion of the treatment device within a kit is alsoviewed as a significant improvement over U.S. application number US2014/0234212 in which the treatment device alone is disclosed due to thesafety and efficacy reasons described above.

In representative embodiments, the kit includes a mucoadhesive drugdelivery device containing active agent-encapsulating microparticles anditems useful for the successful administration and disposal of thedevice, such as: an oral permeation enhancer either incorporated withinthe delivery device or provided within the kit alongside it, and an oralrinse used to cleanse the mouth prior to or following treatment.

FIG. 7 is a schematic depiction of a kit containing 6 treatment devices,where: 71 refers to individual containers of permeation enhancers (ineither solution or powder form), one applied for each treatment, 72forceps or forceps-like instrument used to apply the treatment device,73 cotton/sponge tipped applicator used to apply the permeationenhancer, 74 gauze or other absorbent pad to be used during treatment,75 container of mouth rinse, some of which will be used during eachtreatment, 76 Disposable packages for the remainder of device and otherwaste generated during application, 77 identifies the structural sideswithin the kit that separate the materials from one another, 78 sixindividually packaged and stored treatment devices.

FIG. 8 is an image taken of one of the treatment kits, designed inaccordance with the depiction of FIG. 7.

FIG. 9 is another schematic representation of a kit, which includes: 91one container of permeation enhancer which is utilized during all 6treatments (in either solution or powder form), 92 the oral mouth rinseused during each treatment, 93 gauze pad or other absorbent materialused during treatment, 94 disposable forceps or forceps-like devicesused during treatment to place and remove the treatment device, 95multiple disposable applicators used to apply the permeation enhancerduring each treatment, 96 Disposable packages for the remainder ofdevice and other waste generated during application, 97 identifies thestructural sides within the kit that separate the materials from oneanother, 98 six individually packaged and stored treatment devices.

Targeted Gastrointestinal Delivery of Agents

In another aspect, a disclosed particle-based agent delivery system hasbeen developed for the treatment of gastrointestinal diseases andconditions. The delivery device disclosed herein is in part effectivebecause of its ability to adhere to the intestinal mucosa.

The properties of mucus itself must first be understood to properlydevelop a delivery system. Mucus is a viscoelastic gel layer thatprotects tissues that would otherwise be exposed to the externalenvironment. Mucus is composed primarily of crosslinked and entangledmucin fibers secreted by goblet cells and submucosal glands. Mucins arelarge molecules, typically 0.5-40 MDa in size, and coated with a complexand highly diverse array of proteoglycans. Mucus pH can vary greatlydepending on the mucosal surface, with highly acidic environmentscapable of aggregating mucin fibers and greatly increasing the mucusviscoelasticity. In the human GI tract, the mucus layer is thickest inthe stomach and the colon. Gastric mucus is exposed to a wide range ofpH: a large pH gradient exists within the same mucus cross-section, withpH rising from the luminal pH of 1-2 to 7 at the epithelial surface.

Accordingly, provided herein is a gastrointestinal drug delivery systemcapable of modulating the release of agents depending on theenvironmental pH, thereby making possible the specific targeting ofdrugs within the gastrointestinal tract. Described herein are efforts totarget drug delivery to the gastrointestinal tract through the design ofa mucoadhesive delivery system which releases its payload only withinthe pH environment of the gastrointestinal (GI) tract, and, morespecifically, to specific regions within the GI tract. Because of themucous lining of the GI tract, attraction to the mucosa andmucoadhesivity are important elements of this device.

In the case of orally administered agents for gastrointestinal delivery,survival through digestive regions of extreme pH values is necessary. Inthe human stomach, the volume of gastric fluid ranges from 20 to 100 mlwith a pH of 1.5-3.5. Gastric fluid consists of hydrochloric acid,potassium chloride and sodium chloride. Fluid secretion takes place overseveral stages. Hydrogen and chloride ions are secreted and mixed in thecanaliculi. The lumen of the oxyntic gland secretes the gastric acidwhich reaches the stomach lumen. Secretion of the chloride and sodiumions creates a negative potential of approximately −35 to −65 mV, whichallows for diffusion of the potassium and sodium ions from thecytoplasm.

Carbonic anhydrase forms carbonic acid by catalyzing reactions betweenwater and carbon dioxide. This allows for the dissociation into hydrogenand bicarbonate ions. The hydrogen ions then move from the cell. Sodiumions are reabsorbed. In the canaliculus, hydrogen and chloride ions mixand are secreted into the lumen of the oxyntic gland.

Gastric acid production is separated into three phases. The first ofthese is the cephalic phase, where approximately 30% of gastric acidproduction is stimulated by the smell, taste, or expectation of food assignaled by the brain. About 50% of gastric acid is produced during thegastric phase, where stimulation of production occurs by food presencein the stomach and release of amino acids from consumed materials. Theintestinal phase represents the last phase of acid production, where theremaining 20% of acid is produced when chyme (semifluid of partiallydigested food) enters the small intestine. If agents targeted to thegastrointestinal mucosa are to be delivered orally, the delivery systemmust remain intact and stable through the gastric acid of the stomach.Through the development of a system which is able to remain stable inthis environment as well as multiple pH conditions, oral administrationfor delivery of an agent into and within the gastrointestinal mucosa ismade possible. As opposed to traditional products with an entericcoating, this system is both able to withstand a multitude of pH levelsas well as contain a combination of particles which are able to releasein desired pH conditions.

In order for orally administered alternatives to enteric capsules to beefficacious in the treatment of gastrointestinal diseases, suchalternatives should possess the capability of remaining stablethroughout the entire described acidic and dynamic stomach digestionprocess. Likewise, since many GI diseases may encompass multiple regionsof the GI tract that span multiple pH ranges, the development of adelivery system that is able to withstand and release over various pHchanges is advantageous. An efficacious alternative should also becharacterized by the ability of becoming attracted by means ofmucoadhesivity to the intestinal mucosa lining and releasing uponcontact or at a designated time thereafter. Thus, provided herein is atherapeutic, diagnostic and/or prophylactic delivery device for localand systemic administration and delivery into the gastrointestinal stemcells and/or systemically beyond, which is able to becomeattracted/attach to and penetrate through the intestinal mucosa as wellas remain stable in acidic stomach conditions. The devices describedherein are able to provide an extended or delayed release, programmablerelease, and site specific release into and within gastrointestinallocations.

In many instances, oral administration is not possible if the patient isunable to swallow a capsule or tablet. This can occur with youngchildren where compliance is low or among the elderly where pain existsor there is otherwise an inability to take oral medication. People withfeeding or nasogastric tubes are other examples of these cases. It istherefore an additional object of some embodiments of the presentinvention to provide a method of oral delivery of agents to patients whootherwise would be unable to be administered oral agents. This is inpart accomplished through the optional use of liquid and gelatin formsof oral administration for those who cannot swallow solid tablets orcapsules, as well as through nasal administration or consumption througha nasogastric or feeding tube. Compared to traditional administrationtechniques, the delivery system provides for the successful deliverythrough theses avenues. Particles may be provided in a variety of formsand tailored to specific needs.

The gastrointestinal delivery system also provides a therapeutic,diagnostic and/or prophylactic delivery device that is effective in thepresence or potential presence of gastrointestinal fluids, in contrastto the traditional washout problems described above associated with suchfluids. Also provided is a route for administering a therapeutic,diagnostic and/or prophylactic agent to one or multiple specific regionsof the intestinal epithelia through the design of a system containingone or more sets of particles able to withstand and release through amultitude of pH ranges.

For these and other purposes, the gastrointestinal delivery systemdisclosed herein may serve as a device for specific, targeted deliverywithin the gastrointestinal tract to the gastrointestinal mucosa. Inexemplary embodiments, the delivery device adheres to thegastrointestinal mucosal tissue, is able to withstand the low pH, acidicenvironment of the stomach and contains pH-targeted, mucoadhesiveparticles in which one or more agents are encapsulated. The device mayalso include permeation enhancers sufficient to facilitate agentpermeation through the mucosal layer of the gastrointestinal tract.

One of the unique properties of this platform is that the release andtargeting attributes can be controlled based on desired parameters. Theparticles may be controlled to remain stable within a desired pH leveland release in another desired pH level. This ability may be used tocreate a combination of targeted particles which can remain stablethrough any component of the GI tract regardless of pH exposure,including the stomach, esophagus, and components of the intestines.

In addition to stability, the timing of release of the agentsencapsulated within the particles may be controlled. The purpose of thisfeature is to further target the delivery to locations within the GItract. For example, if it is known that under normal digestionconditions, a known amount time is known to intercur between oralconsumption and delivery to the desired delivery, the particles withinthe delivery system may be further designed to release their agentpayload at such amount of time after oral contact is made with thedelivery system. A formula has been developed by which the release timemay be determined. The parameters and formula are identified below:

-   -   Degree of Deacetylation (DA)    -   Molecular Weight (MW) combinations,    -   Time (T)    -   Amount of exposure to humidity, water content (WC)    -   Solution pH (SpH) at the synthesis stage    -   Degree of Viscosity (DV)    -   Synthesis technique (K is a constant) such as freezing method of        the particles

Degree of release(DR)=a(DA)+b(MW)+c(SpH)+d(T)+e(WC)+f(DV)+k  Formula:

** It has been discovered that the use of sodium nitrite allows forfurther modulation of the degree of deacetylation and molecular weightof chitosan.

The pH modulation and configurable release timing increases the efficacyof the delivery system and shows how innovative it is compared totraditional systems. Without wishing to be bound to any particulartheory, it is believed that such superior properties are the product ofpreviously unknown effects of the degree of deacetylation and molecularweight of the chitosan. FIG. 10 shows the pH levels of a portion of theGI tract. Using this information and the relationship between pH andlocation, delivery can be targeted. For example, particles can beprogrammed to release at pH levels of only between 5.8 and 6.2, therebymaking possible the specific targeting to the duodenum.

Targeting release to multiple locations can be achieved by the inclusionof a blended mix of customized particles within the delivery system. Anexample of this application is shown in FIG. 11. This allows fortargeting to an array of desired locations if a disease or condition islocated in multiple regions, or if an agent is best delivered over arange of locations.

I. Agent Delivery Device

The device includes agent-encapsulating particles consisting of apolymer having dispersed or encapsulating therein a therapeutic,prophylactic, diagnostic or nutraceutical agent (FIG. 13). If desired,the particles may include chemical linkers, which can couple targetingligands and/or additional agents to the particles (FIG. 13). The deviceincludes these particles as well as preferably permeation enhancers.

The device may be orally administered via tablet, capsule, liquid,syrup, gelatin or other oral consumable, or via nasogastric tube orfeeding tube for those who are unable to swallow, and exhibitsproperties which allow the agent encapsulated in the particles to remainstable in the variably acidic environment of the stomach. The device isable to deliver agent-encapsulated particles (“loaded particles”, alsolabeled as “LPs”) to the epithelial cells within the gastrointestinal(GI) tract (FIG. 12). The particles adhere to the intestinal mucosa anddegrade, releasing the encapsulated agent directly into the intestinalepithelium. FIG. 16 illustrates the drug delivery process.

The particles (preferably having an average diameter of 500 to 2000nanometers (nm)) permeate the mucosal tissue of the intestine (FIGS. 14and 15 and are taken up by intestinal stem cells (ISC's). This size isadequate to carry enough therapeutic, diagnostic or nutraceutical agentto obtain high loading and encapsulation efficiencies (higher than 80%),which is desirable for scaling up and commercialization.

The encapsulation of therapeutic, diagnostic and/or nutraceutical agentsallows for controlled penetration into the mucus, by means of adding atargeting ligand, as well as a controlled release profile. Moreover, inthe case of systemic penetration, the encapsulation also reduces uptakeby the body's reticuloendothelial system. The smaller particles havegreater surface area-to-volume ratios, which cause the particles'dissolution rates to be higher than that of larger particles.

Many agents are limited in delivery due to solubility factors. The largesurface area to volume of particles increases the bioadhesivity. Thesefactors in combination result in the penetration of the agent deep intoISCs, providing a greater benefit (FIGS. 14 and 15).

There are three main aspects of targeting that work to achieve localizeddelivery:

1. Charge: Positively charged polymers may used in the synthesis of theparticles included in this delivery system. The resulting deviceexhibits a positive charge which attracts the agent-encapsulatedparticles to the negatively charged mucosa of the intestine.

2. Activity: Obtained using molecular targeting agents to further focuson ISCs.

3. pH: Particles in the present compositions are able to be modified toremain stable or release agents in varying pH environments, asillustrated in Example 3 under Methods of Administration. Thisdetermination takes place during the synthesis process. By synthesizingparticles which keep the agent encapsulated in the highly acidicenvironment of the stomach and promote release in the more basicenvironment of the intestines, release is therefore targeted. Theseparameters can be changed to include release in more acidic environmentsor combinations thereof (FIG. 22B). These parameters can also be changedto allow for release in both the stomach and intestines. Further,varying doses and varying drug combinations are also possible. FIG. 22Cshows an example of particles which release as the pH approaches neutrallevels.

A. Mucoadhesive Polymeric Particles

Several bioadhesive and mucoadhesive polymers are known. In thepreferred embodiment of this concept, the polymer is mucoadhesive sothat it can bind to the mucosal intestinal tissue. Preferably, thepolymer is polycationic, biocompatible, and biodegradable. The preferredpolymer is chitosan. Chitosan is a polycationic, non-toxic,biocompatible and biodegradable polymer. Chitosan is commonly used as amucosal agent delivery mechanism because of its bio-adhesiveness andpermeability properties. The barrier in GI epithelium can easily bedisrupted by chitosan particles, enhancing permeability through themucosa.

Different factors affect fabrication of chitosan particles, such as pHof the preparation, inclusion of polyanions, charge ratios, the degreeof deacetylation and the molecular weight of chitosan.

Chitosan particles have, to date, been preferred for use withchemotherapeutics for cancer treatment because of the chitosan'ssensitivity to low pH. Since cancer tissue is acidic, chitosan particlesrelease the agent faster in an acidic environment. However, in contrastto traditional uses of chitosan particles, provided herein is a novelchitosan particle synthesis process to allow for stability in acidicenvironments and agent release when exposed to basic conditions. Thecontrolled release of the agent from the chitosan particles ensures thata steady amount of agent penetrates the proper GI mucosal tissue whileminimizing loss and exposure to fluids and other tissue.

B. Agents to be Encapsulated

Any therapeutic, prophylactic, diagnostic or nutraceutical agent whichis capable of encapsulation and release within the GI tract may be used.Representative agents include biologics, peptides, nucleotides,anti-infectives, antibiotics, antifungals, antivirals,anti-inflammatories, immunomodulators, vaccines, and combinationsthereof. Preferred agents include calcium mobilizers such as nicotinicacid adenine dinucleotide phosphate and peptides such as glucagon-likepeptide-2. Nicotinic acid adenine dinucleotide phosphate and othercalcium mobilizers have been shown to promote ISC proliferation andintestinal epithelium regeneration. Glucagon-like peptide-2 and itsanalogs have also been shown to promote ISC proliferation and intestinalepithelium regeneration. The efficacy of nicotinic acid adeninedinucleotide phosphate and glucagon-like peptide-2 have been shown to behindered by a lack of proper targeting and delivery within the GI tract.The particle-based system provided herein remedies such drawbacks.

C. Additional Additives

Other compounds which may be added include permeation enhancers andantioxidants that are useful to prevent bacterial contamination. Theseagents may be coated onto, encapsulated within or mixed among theagent-encapsulating particles.

Methods of Manufacture

There are at least four methods available to make chitosan particles:ionotropic gelation, microemulsion, emulsification solvent diffusion andpolyelectrolyte complex. The most widely developed methods areionotropic gelation and self-assembling polyelectrolytes. These methodsoffer many advantages such as simple and mild preparation conditionswithout the use of organic solvents or high shear forces. They areapplicable to broad categories of agents including macromolecules whichare notorious as labile agents. It has been found that the factors foundto affect particle formation, such particle size and surface charge,also include the molecular weight and degree of deacetylation of thechitosan. The entrapment efficiency has been found to be dependent onthe pKa and solubility of entrapped agents.

The ionotropic gelation method is commonly used to prepare chitosanparticles. In an acidic solution, the amine group of chitosan moleculesis protonized and interacts with an anion such as sodiumtripolyphosphate (STPP) by ionic interaction to form particles (Lee, etal., Polymer, 42:1879-1892 (2001)). This method is very simple and mild.Reversible physical crosslinking by electrostatic interaction, insteadof chemical crosslinking, may be applied to prevent possible toxicity ofreagents and other undesirable effects (Shu, et al., Internal. J Pharm.,201:5158(2000)).

To improve the loading efficiency (LE), a 0/W/0 (oil/water/oil) doubleemulsion method was combined with temperature-programmed solidificationtechnique and controlled PTX within the matrix network as in situnanocrystallite form. Furthermore, these CMC particles were PEGylated,which could reduce recognition by the reticuloendothelial system (RES)and prolong the circulation time in blood. Methods of making chitosanparticles by microemulsion are also known. For example, an amphiphilicgraft copolymer using chitosan as a hydrophilic main chain andpoly(lactic-co-glycolic acid) (PLGA) as a hydrophobic side chain isprepared through an emulsion self-assembly synthesis. A chitosan aqueoussolution is used as a water phase and PLGA in chloroform serves as anoil phase. A water-in-oil (W/O) emulsion is fabricated in the presenceof the surfactant span-80. TIle CS-g-PLGA amphiphile can self-assembleto form micelles with size in the range of 100-300 nm, which makes iteasy to apply in various targeted-drug-release and biomaterial fields.Chitosan can be dissolved into deionized water together with1-hydroxybenzotriazole. A water-in-oil (W/O) chitosan andpoly(lactic-co-glycolic acid) microemulsion is prepared and then achitosan-graft-poly(lactic-co-glycolic acid) stimuli-responsiveamphiphile is fabricated. The obtained amphiphile can self-assemble toform micelles. In suitable solvents. Cai, Int J Nanomedicine, 6:3499-508(2011), describes RGD peptide-mediated chitosan-based polymeric micellestargeting delivery for integrin-overexpressing tumor cells.

The cationic amino groups on the C2 position of the repeatingglucopyranose units of chitosan can interact electrostatically with theanionic groups (usually carboxylic acid groups) of other polyions toform polyelectrolyte complexes. Many different polyanions from naturalorigin (e.g. pectin, alginate, carrageenan, xanthan gum, carboxymethylcellulose, chondroitin sulphate, dextran sulphate, hyaluronic acid) orsynthetic origin (e.g., poly (acrylic acid)), polyphosphoric acid, poly(L-lactide) have been used to form polyelectrolyte complexes withchitosan in order to provide the required physicochemical properties forthe design of specific drug delivery systems (Berger et al. Eurl PharmBiopharm. 2004; 57:35-52).

III. Methods of Administration

The particles encapsulating one or more agents may be administered viaenema, capsule, solid, liquid or combination, either orally, vianasogastric tube or feeding tube, or rectally via endoscopic instrumentssuch as an endoscope.

Example 1: Preparation of Programmable Chitosan Particles

Chitosan particles (“Particles”) at varying pHs were prepared using USPgrade chitosan, as described below. The particles were then subjected toincremental pH adjustments using sodium hydroxide or hydrochloric acid.The size at synthesis after each adjustment was recorded.

Impact of pH Increase

Chitosan (95% degree of deacetylation, 50k MW) (CHI) was dissolved in0.175% acetic acid to a final concentration of 0.2% (w/v). Sodiumtripolyphosphate (STPP) was dissolved in deionized water to a finalconcentration of 0.2% (w/v). Particles were synthesized via the ionicgelation technique, which involves the methodical addition of anelectrostatic cross-linker (TPP) to an aqueous polymer solution (CHI).The pH of the CHI solutions were adjusted with 0.1 M sodium hydroxide(NaOH) or 0.1 M hydrochloric acid (HCl) accordingly (1.0, 2.0, 2.25,2.50, 2.75, 3.0, 4.0, 5.0 and 6.0).

Briefly, 40.0 mL of each CHI solution was added to a beaker undermagnetic stirring. To this, 10.0 mL of TPP solution was added drop-wiseat a constant flow rate. The Particles were allowed to stabilize for 1hour and initial size measurements were taken using a Zetasizer Nano(Malvern Instrument Ltd., UK). The pH of the Particles was increasedusing 0.1 M NaOH at increments of 0.5 or 1.0 depending on the initialpH. At each step, the size and polydispersity index (PDI) were recorded.

Impact of pH Range Fluctuations

Chitosan (95% degree of deacetylation, 50k MW) (CHI) was dissolved in0.175% acetic acid to a final concentration of 0.2% (w/v). Sodiumtripolyphosphate (STPP) was dissolved in deionized water to a finalconcentration of 0.2% (w/v). Particles were synthesized via the ionicgelation technique, which involves the methodical addition of anelectrostatic cross-linker (TPP) to an aqueous polymer solution (CHI).The pH of the CHI solutions were adjusted with 0.1 M sodium hydroxide(NaOH) or 0.1 M hydrochloric acid (HCl) accordingly (1.0, 2.0, 3.0, 4.0,and 5.0).

Briefly, 40.0 mL of each CHI solution was added to a beaker undermagnetic stirring. To this, 10.0 mL of STPP solution was added drop-wiseat a constant flow rate. The Particles were allowed to stabilize for 1hour and initial size measurements were taken using a Zetasizer Nano(Malvern Instrument Ltd., UK). The pH of the Particles wereincreased/decreased using 0.1 M NaOH or 0.1 M HCl at increments of 1.0on a scale of 1.0-7.0, depending on the initial pH. The purpose of thisstudy was to simulate the rapid and drastic pH changes of the GI tractif Particles were given orally to a patient. At each step, the size andpolydispersity index (PDI) were recorded.

Impact of Polymer State

Chitosan chloride (95% degree of deacetylation, 50k MW) (CHI) wasdissolved in 0.175% acetic acid to a final concentration of 0.2% (w/v).Sodium tripolyphosphate (STPP) was dissolved in deionized water to afinal concentration of 0.2% (w/v). Particles were synthesized via theionic gelation technique, which involves the methodical addition of anelectrostatic cross-linker (STPP) to an aqueous polymer solution (CHI).The pH of the CHI solutions were adjusted with 0.1 M sodium hydroxide(NaOH) or 0.1 M hydrochloric acid (HCl) accordingly (1.0, 2.0, 3.0, 4.0,& 5.0).

Briefly, 40.0 mL of each CHI solution was added to a beaker undermagnetic stirring. To this, 10.0 mL of STPP solution was added drop-wiseat a constant flow rate. The Particles were allowed to stabilize for 1hour and initial size measurements were taken using a Zetasizer Nano(Malvern Instrument Ltd., UK). The pH of the Particles wereincreased/decreased using 0.1 M NaOH or 0.1 M HCl at increments of 1.0on a scale of 1.0-7.0 depending on the initial pH. The purpose of thisstudy was to simulate the rapid and drastic pH changes of the GI tractif Particles were given orally to a patient. At each step, the size andpolydispersity index (PDI) were recorded. This study served to show thesensitivity of pH changes of chitosan vs. its salt derivative.

Effect of Molecular Weight & Degree of Deacetylation

Chitosan (95% degree of deacetylation, 50k MW) (CHI) was dissolved in0.175% acetic acid to a final concentration of 0.2% (w/v). Sodiumtripolyphosphate (STPP) was dissolved in deionized water to a finalconcentration of 0.2% (w/v). To the chitosan, 200 μL of 60 mM sodiumnitrite solution was added and the mixture stirred for 15 minutes.Without being bound to any particular theory, it is believed that thisserved to lower the molecular weight and the degree of deacetylation ofchitosan by chemically breaking the 1,4 glycosidic bonds of the polymer.Particles were synthesized via the ionic gelation technique, whichinvolves the methodical addition of an electrostatic cross-linker (STPP)to an aqueous polymer solution (CHI).

Briefly, 40.0 mL of each CHI solution was added to a beaker undermagnetic stirring. To this, 10.0 mL of STPP solution was added drop-wiseat a constant flow rate. The Particles were allowed to stabilize for 1hour and initial size measurements were taken using a Zetasizer Nano(Malvern Instrument Ltd., UK). The pH of the Particles wereincreased/decreased using 0.1 M NaOH or 0.1 M HCl at increments of 1.0on a scale of 1.0-7.0, depending on the initial pH. The purpose of thisstudy was to simulate the rapid and drastic pH changes of the GI tractif Particles were given orally to a patient. At each step, the size andpolydispersity index (PDI) were recorded. Results of this and otherstudies are shown in FIGS. 23, 24 and 25.

Results:

FIGS. 23, 24, and 25 summarize results of conducted studies includingthe properties of the particles prepared from low molecular weightchitosan. By far, the most important physical property of particulatesamples is particle size. Measurement of particle size distributions isroutinely carried out across a wide range of industries and is often acritical parameter in the manufacture of many products. Preferably, theparticles have an average diameter of from about 500 nm to about 2000nm.

As described in Heurtault, et al., Biomaterials, 24:4283-4300 (2003),Zeta potential is an important and useful indicator of particle surfacecharge, which can be used to predict and control the stability ofcolloidal suspensions or emulsions. Almost all particles in contact witha liquid acquire an electric charge on their surface. The electricpotential at the shear plane is called the zeta potential. The shearplane is an imaginary surface separating the thin layer of liquid(liquid layer constituted of counter-ions) bound to the solid surface inmotion. The greater the zeta potential the more likely the suspension isto be stable because the charged particles repel one another and thusovercome the natural tendency to aggregate. The measurement of the Zetapotential allows predictions to be made about the storage stability of acolloidal dispersion.

The charge of the particles is important for their mucoadhesivenessproperty (Biol Phann Bull. 2003 May; 26(5):743-6. Positively chargedmaterials have better mucoadhesion strength likely due to the negativecharge of mucosa. Therefore, the required positive charge is within arange of 10-50 mV, which is regarded as stable.

In a separate experiment, particle were also prepared as describedabove, using pharmaceutical grade chitosan: PROTASAN™ UP CL 113, acommercially available formulation of chitosan chloride (FMCCorporation, Pennsylvania). PROTASAN™ UP CL 113 is based on a chitosanwhere between 75-90 percent of the acetyl groups are removed. Thecationic polymer is a highly purified and well-characterizedwater-soluble chloride salt. The functional properties are described bythe molecular weight and the degree of deacetylation. Typically, themolecular weight for PROTASAN™ UP CL 113 (chitosan chloride) is in the50,000-150,000 g/mol range (measured as a chitosan acetate). Theultra-low levels of endotoxins and proteins allow for a wide variety ofin vitro and in vivo applications.

The graphs of FIG. 23A-23C show the properties of certain particles indifferent situations, including FIG. 23A: synthesis pH size vs pHadjustment; FIG. 23B: pH change to size; FIG. 23C: pH change topolydispersity index (PDI). The graphs of FIGS. 24A-24D illustrate thesensitivity of one selected loaded particle (“LP”) variant (FIGS. 24Aand 24B) as well as the level of modulation and control that can be heldover particle properties (FIGS. 24C and 24D). The charts of FIGS.25A-25D demonstrate an example of how the size (FIGS. 25A and 25D) andcharge (FIGS. 25B and 25C) of chitosan loaded particles can be modulatedusing different concentrations of preprocessing agents such as sodiumnitrite.

By using high purity chitosan (the cationic polymer was a highlypurified and well-characterized water-soluble chloride salt), chitosanparticle of better quality were obtained. The optimal formulation wasobtained by optimizing different ratios between the STPP and chitosansolution (0.6:1; 0.5:1; 0.4:1). The best formation was achieved with aratio of STPP to chitosan of about 0.5:1, which had the ideal size andzeta potential.

Example 2: Effect of pH on the Size, Zeta Potential and Stability of

chitosan particle. This technology is advantageous because of itsability to be customized to remain stable in desired pH environments andrelease in other desired pH environments. This is accomplished in partby modulating the pH during the particle synthesis process. An exampleof the effect of the synthesis pH on the release of particle issummarized in FIG. 26, with the effect on size and Polydispersity Index(PDI) shown.

As shown in FIGS. 26A-26E, the polydispersity index (PDI) and size ofthe particle change depending on the pH environment introduced aftersynthesis, as seen by comparing the results obtained at pH 3.4 (FIG.26A), pH 1 (FIG. 26B), pH 2 (FIG. 26C) pH. For this example, a PDI ofapproximately 0.40 or lower is preferable. Since the polymer making upthe particles used above expands to release the encapsulated drug, alarger size indicates that the particles have degraded and released.

As shown in FIG. 27, the pH of the human intestines range dramatically,and drug delivery systems which are able to be customized to controlrelease according to pH are highly effective at targeting. For example,according to the example data shown above, Particles synthesized at pH 1are able to remain stable from pH range 3-5 and release in a lower orhigher environment. A patient that is administered these particlesalongside some other supplement would experience targeted release withinthe ileum. Likewise, other formulations can yield particles that canpromote delivery all across the GI tract.

In another embodiment, particles have been designed to remain stable atpH levels of 2-4 and release at environments above pH 4. FIG. 20 andFIG. 21 illustrate this. FIG. 20 plots the dependency of the size ofparticles (as measured by a Malvern Zetasizer Nano) on the pH. The sizeis seen increasing sharply at levels at and above pH 4. When theparticles release the encapsulated agent, they swell in size, so thisincrease indicates that the agent has been released. Likewise, FIG. 21plots the pH to PDI of the particles.

In another embodiment, particles with different stability and releaseproperties are combined in one delivery method to facilitate delivery tomore than one location within the GI tract despite differences in pH.FIG. 27 illustrates the design of this system.

As shown in FIG. 27, the inclusion of particles with differentproperties in the delivery medium allow for delivery of particles andsubsequent degradation within various regions of the GI tract. This isadvantageous over traditional methods of agent delivery due to itsenhanced targeted ability and non-reliance on merely one “stable” pHrange and where the particles release encapsulated agent.

Example 4: Optimization of NP at Large Scale

Automation of processes is especially necessary in nanotechnologyapplications where the product properties are very sensitive to changes.

Materials and Methods for automated particle synthesis:

All the reagents and chemicals used are excipient or pharmaceuticalgrade

Solution A: 0.1% (active agent) in 0.1% Tripolyphosphate (STPP) solution

Solution B: 0.1% Chitosan (CL 113) in 0.175% acetic acid solution

Place 10 mL solution B in a glass beaker and stir at 600 rpm on magneticstirrer. Transfer a total of 10 mL Solution A drop wise on the stirredsolution B with the help of a peristaltic pump or any other pump thatcan provide a constant flow rate, as used herein at 1.5 mL/min, butwhich may be modified to yield a different size, charge, polydispersity,NP yield, and drug encapsulation efficiency properties.

Different ratios of solutions A to solution B (A:B) were used from 1:1(as in the above case) to 1.1:0.85. Different mixing ratios producedifferent NP properties. Gradually increase the stirring speed ofsolution B to 650 rpm at the time when half of the solution A has beentransferred. When transfer of solution A is completed, graduallyincrease the stirring speed to 700 rpm and then gradually adddisaccharide trehalose to the solution to obtain a final trehaloseconcentration of 2%. Continue stirring until all the added trehalose hasbeen dissolved (or for at least 10 min) to equilibrate the solution.Measure the Z-average, the polydispersity index (PDI), the mean diameterof each peak, and NP yield (count rate) of the obtained Particles. Thefollowing storage steps are followed.

For storage, the final NP solution is placed in a proper container andis frozen using liquid nitrogen, in dry ice, or in ultra-low temperaturefreezer until complete freezing obtained and then they are freeze-drieduntil complete elimination of solvent is obtained.

NP Solution

Scalability is essential for mass production. Drug carrying particlesare formed in a solution environment by self-assembly, which is adynamic process that takes place only under the correct chemicalconditions. This technique is extremely sensitive to manufacturingvariables including mixing rate of subsequent solutions and theirconcentrations, freshness, and purity. The mixing rate of subsequentsolutions cannot be increased above a certain value without sacrificingparticle properties. Furthermore, due to the dynamic nature ofself-assembly, the mixing process must be finished in a limited time asany delays in this duration increases the chance of deviation ofparticle properties or yield from optimal values. The sensitive natureof self-assembly production methodologies enforces adoption of strictlycontrolled batch type manufacturing processes and does not allow highproduction volumes per batch. An automated system has been developedwhich has successfully adapted the original manual particle productionprocesses into an automated version and can now strictly control theproperties of the resulting particle including size, polydispersity, andencapsulation efficiency. These parameters are of prime importance interms of efficacy of the drug delivery system as well as raw materialcosts. Parameters can be adjusted to obtain a balance between maximalbatch volume and particle yield (number of particles formed in aparticular production batch) while keeping the properties of particleswithin narrow tolerances. Polydispersity of the obtained particles wasfound to be well within acceptable ranges.

Targeted Delivery of Agents for the Treatment of Colorectal and AnalDiseases

In a further aspect, a drug delivery device has been developed toadminister agents in a highly targeted manner within the colonic,rectum, and/or anal tissue. This device is in the form of a mesh, ormesh-like material, and is topically applied onto the anus, perianalarea or through the anal canal onto affected tissue in need of agentdelivery. The mesh releases agents encapsulated within microparticlesfor controlled and targeted treatment of diseases. The mesh is able todeliver microparticles through the mucosa or other tissue with which itcomes in contact, to deliver a concentrated dosage of agents locally, ina more targeted manner than traditional systemic delivery of agents.This allows for greater safety and efficacy of agents within the body.The microparticles included within the mesh provide retention of theagent or agents locally and/or regionally within the tissue to which themesh was applied. If desired, free agents within the mesh but not withinthe particles may also be included, for example to achieve agentdelivery from the local tissue into systemic circulation of the body.

The mesh formulation utilizes microparticles to target drug delivery andalter release parameters which can treat or prevent diseases, orotherwise deliver agents to the colon, rectum, or anal tissue to treat,prevent and/or diagnose diseases. The use of microparticles allowspassage through the mucosa lining the tissue of the colon, rectum,and/or anus, ensuring a sufficient and targeted agent delivery.

The formulation of this mesh includes agent-encapsulatingmicroparticles. The properties of these microparticles are able to bechanged to modulate the release depending on the region of thegastrointestinal tract being treated. For example, microscopically, therectum is lined by high columnar mucus-producing cells. The anal canalis divided into three zones with different type of epithelium lining;the proximal zone is lined by stratified cuboidal epithelium, while theintermediate (pectin zone) is lined by stratified squamous epitheliumbut without adnexae (e.g., hair, sebaceous glands). And the distal (theanal skin) is lined by squamous stratified epithelium and contains hairand sebaceous glands. To this end, different sizes of particles may berequired in order to permeate and release the encapsulated agent withinthe specific affected region, maximizing the specificity of thedelivery. The size and charge of the particles may be customized toprovide for the adequate treatment of each region. This further allowsfor the development of an optimal personal treatment customized for thelocation and disease.

The microparticles are formed of a polymer such as chitosan, and willinclude at least one agent to be delivered. This delivery system mayoptionally include permeation enhancers, either included within the meshor externally administered to the targeted tissue prior to, during, orafter mesh administration. Any permeation enhancer or permeationenhancers administered will affect at least the tissue of the colon,rectum, and/or anus. They may be administered via enema, liquid,suppository, orally administered capsule, tablet, injection, within themesh itself or via other delivery means.

In the preferred embodiment, the mesh is formed with one side exposedfor contact with the appropriate tissue. As illustrated in FIG. 28,particles containing the agent or agents will be released from this sideupon contact with the appropriate tissue. Also in the preferredembodiment, the other side facing the external gastrointestinal cavitymay be covered with a backing film, layer, or other impermeable membraneto prevent loss of the agent from the mesh into the gastrointestinalsystem, or contamination of the mesh via fluids or other matter in thegastrointestinal system. Optionally, the impermeable film, layer, ormembrane may be replaced with a water-permeable film, layer, ormembrane.

The mesh described herein can also be included as one component of alarger treatment kit intended for the treatment of colon, rectal and/oranal diseases. This kit may include materials that are required forproper administration of the mesh as well as proper and safe disposal ofthe mesh after application and cleaning of the treated area. Forexample, 5-fluorouracil, mitomycin and cisplatin are commonly usedchemotherapeutics for the treatment of anal cancer. And either theFOLFOX (5-FU, leucovorin, and oxaliplatin) or CapeOx (capecitabine andoxaliplatin) regimens are used most often in treating colorectal cancer.These chemotherapeutics are highly potent and toxic, however, andextensive precautions must be taken to ensure that proper handlingprocedures are followed during treatment, and contact is minimizedbetween these agents and both the patient and personnel applying themesh. Items that may then be included in the kit for the purpose ofsafety, such as a cleansing enema to be administered before and/or afterapplication of the mesh, forceps or other tools for the placement of themesh, disposable packaging for any remaining portion of the mesh afterapplication, and any other instruments which may be required to safelyadminister the mesh.

A topical agent delivery mesh has been developed specifically fordelivery of agents within the rectal, colonic, and/or anal tissue.Preferably, the mesh is at least partially made of biodegradable and/orbiocompatible materials. The microparticles released from the mesh arepreferably locally or regionally retained within the colonic, rectal,and/or anal tissue under and around which the mesh was applied. Themajority of the one or more agents released from the microparticles ispreferably locally or regionally retained within the colonic, rectal,and/or anal tissue under and around which the mesh was applied.

The Mesh:

The mesh includes microparticles and an optional backing layer fordelivery through mucosa, epithelium, or both, and deliversmicroparticles into neighboring tissue (FIG. 28). The mesh can takemultiple forms, typically synthesized within a mold to control its shape(FIG. 29). Microparticles can be seen within the mesh (FIG. 32) and helpto form its structure. It can be seen that microparticles offersignificant contribution to the mesh structure. The microparticlesinclude a polymer, and optionally targeting elements (FIG. 13). The meshis applied to the tissue of the gastrointestinal tract via the anus,preferably onto a mucus layer but also onto epithelium of the anus whereno mucus is present, allowing delivery of the agent to the underlyingcells. The microparticles penetrate the mucosa/epithelium and releasethe agent or agents directly into the tissue. The mesh may be formed ofbioadhesive polymer.

In the preferred embodiment, chitosan is used as the polymer for themicroparticles. Chitosan is a deacetylated derivative of chitin, thesecond most abundant polysaccharide, and has a large density of reactivegroups and a wide range of molecular weights. Chitosan is considereduseful as a bioadhesive material because of its ability to formnon-covalent bonds with biological tissues, mainly epithelia and mucousmembranes. Bioadhesions formed using natural polymers have uniqueproperties as a carrier because they can prolong residence time and,therefore, increase the absorbance of loaded drugs. Chitosan is abioabsorbable, biocompatible, biodegradable, anti-bacterial andnon-toxic polymer.

In addition, chitosan has different functional groups that can bemodified. Because of its unique physicochemical properties, chitosan hasgreat potential in a range of biomedical applications. Chitosan can beused as a delivery mechanism because of its bio-adhesiveness as well asits established ability to act as an absorption and permeation enhancer.The barrier in mucosa or epithelium can easily be disrupted by chitosanparticles, enhancing permeability through mucosa.

The most widely developed particle manufacturing methods are ionotropicgelation and self-assembling polyelectrolytes. These methods offer manyadvantages such as simple and mild preparation method without the use oforganic solvent or high shear force. They are applicable to broadcategories of agents including macromolecules which are notorious aslabile agents. Usually, the factors found to affect particles formation,including particle size and surface charge, are molecular weight anddegree of deacetylation of chitosan. The particles may be tailored to bestable in a variety of environments (FIG. 31).

The ionotropic gelation method is commonly used to prepare chitosanparticles. This method is based on electrostatic interaction; atphysiologic pH, the primary amine groups of chitosan are protonated, andtherefore chitosan is positive-charged. The positive charge is used toform particles in solution via cross-linking with polyanions(stabilizer) such as sodium tripolyphosphate (STPP), to efficientlyencapsulate the drug via electrostatic interaction, and to promotecellular internalization of drug-containing chitosan particles. Severaladvantages of this simple and mild method include the use of aqueoussolutions, the preparation of particles with a small size, themanipulation of particle size by the variation in pH values, and thepossibility of encapsulation of drug during particle formation.Structural changes can be introduced by ionic strength variations likepresence of KCl at low and moderate concentrations emphasize swellingand weakness of chitosan-STPP ionic interactions, in turn particledisintegration.

The particles (micro- but also nano-sized particles) can permeate thetissue within the anus, colon, or rectum. The size is dependent on thepH of the solution and the weight ratio of Chitosan to STPP. And thesize of the particles influences the drug release rates. Permeation isimportant to the efficacy of the mesh, and the mesh has been shown tohave very high permeation ability (FIGS. 30 and 33). This size isadequate to carry enough of the included agent or agents such as achemotherapeutic to obtain high loading and encapsulation efficiencies.The encapsulation also protects the outer tissue in the GI tract fromdamage if toxic agents such as chemotherapeutics are included. Otherparameters affect the particles including the chitosan: stabilizer (suchas STPP) ratio in aqueous solution during the synthesis process, as anincrease in the amount of stabilizer leads to a higher degree ofchitosan cross-linking and a decrease in the particle dimensions.

The embodiments of the described above are intended to be merelyexemplary; numerous variations and modifications will be apparent tothose skilled in the art. All such variations and modifications areintended to be within the scope of the present invention as defined inany appended claims.

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What is claimed is:
 1. A system for delivery of a therapeutic agent to asite in mucosal tissue, the system comprising: a porous, mucoadhesive,freeze-dried polymeric matrix, having first and second opposed surfaces,the matrix formed by a composition including chitosan and a plurality ofmicroparticles, having an average diameter between 500 nm and 2000 nm,embedded within the matrix, the microparticles containing a therapeuticagent and having a coating around the therapeutic agent, wherein: (i)the first surface of the matrix is configured to be attached to the sitein the mucosal tissue and the matrix is configured to provide controlledrelease of the microparticles, through the first surface, when the firstsurface of the matrix is thus attached to the site; and (ii) the coatingincludes chitosan so as to provide controlled release of the agent fromthe microparticles.
 2. A system according to claim 1, wherein thecomposition further includes a compound selected from a group consistingof a hydration promoter, a microparticle adhesion inhibitor comprisingHPMC, a microparticle aggregation inhibitor, and combinations thereof.3. A system according to claim 2, wherein the hydration promoter isselected from the group consisting of ethylene glycol, propylene glycol,beta-propylene glycol, glycerol and combinations thereof.
 4. A systemaccording to claim 2, wherein the microparticle aggregation inhibitor isselected from the group consisting of monosaccharides, disaccharides,sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides,and combinations thereof.
 5. A system according to claim 1, wherein themicroparticles further include sodium tripolyphosphate.
 6. A systemaccording to claim 1, further comprising a free amount of thetherapeutic agent, embedded directly in the matrix, and not otherwisecoated with chitosan, wherein the free amount of the therapeutic agentconstitutes between 20-80% of a total amount of the therapeutic agent inthe system.
 7. A system according to claim 1, wherein the second surfaceis permeable to water.
 8. A system according to claim 1, wherein thesecond surface includes a material selected from the group consisting ofa polyacrylate adhesive, a non-woven polyester fabric, and combinationsthereof.
 9. A system according to claim 1, wherein the chitosan in thematrix and the chitosan in the microparticles is pure chitosan.
 10. Asystem according to claim 1, wherein the therapeutic agent is achemotherapeutic pharmaceutical.
 11. A system according to claim 1,wherein the chitosan of the microparticles is pure chitosan.
 12. Amethod for manufacturing a therapeutic agent delivery system, the methodcomprising: forming a first mixture comprising a plurality ofmicroparticles, the microparticles having an average diameter between500 nm and 2000 nm, containing a therapeutic agent and having a coatingaround the therapeutic agent, the coating including chitosan; forming asecond mixture from ingredients including the first mixture andchitosan; freezing the second mixture in a bath containing an aqueousalcoholic solution at a temperature above the freezing temperature ofthe aqueous alcoholic solution and at most −40° C., to form a frozenlayer precursor; and drying the frozen layer precursor, to form a porouspolymeric matrix with microparticles embedded within the matrix.
 13. Amethod according to claim 12, wherein the second mixture furtherincludes a compound selected from the group consisting of a hydrationpromoter, a microparticle adhesion inhibitor comprising HPMC, amicroparticle aggregation inhibitor, and combinations thereof.
 14. Amethod according to claim 12, further comprising embedding a freequantity of the therapeutic agent directly in the matrix, wherein thefree quantity of the therapeutic agent constitutes between about 20-80%of a total quantity of therapeutic agent in the system.
 15. A methodaccording to claim 12, further comprising applying a second layerprecursor to the frozen layer precursor, to form a solid comprising afirst layer and a second layer.
 16. A method according to claim 14,wherein the second layer comprises a therapeutic agent.
 17. A methodaccording to claim 12, further comprising coating the frozen layerprecursor with a frozen second layer precursor, to form a solidcomprising a first layer and a second layer.
 18. A method according toclaim 13, wherein the hydration promoter is selected from the groupconsisting of ethylene glycol, propylene glycol, beta-propylene glycol,glycerol, and combinations thereof.
 19. A method according to claim 13,wherein the microparticle aggregation inhibitor is selected from thegroup consisting of monosaccharides, disaccharides, sugar alcohols,chlorinated monosaccharides, chlorinated disaccharides, and combinationsthereof.
 20. A method according to claim 12, wherein the therapeuticagent is a chemotherapeutic pharmaceutical.